Device for the aseptic expansion of cells

ABSTRACT

A closed system suitable for the aseptic culturing of therapeutic cells. The present system facilitates the aseptic manufacturing of cells for use in therapy, without the need for a clean-room environment because no open processing steps are required.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of and claims priority fromapplication Ser. No. 14/353,170, now U.S. Pat. No. 9,315,774, filed Apr.21, 2014, under 35 U.S.C. § 371 as the U.S. national phase ofInternational Application No. PCT/GB2012/052587, filed Oct. 19, 2012,which designated the U.S. and claims priority from U.S. ProvisionalApplication No. 61/550,246, filed Oct. 21, 2011, each of which isincorporated herein by reference in its entirety including all tables,figures, and claims.

BACKGROUND OF THE INVENTION

The present disclosure relates to an optimised system for asepticallyculturing cells for therapeutic applications on a commercially viablescale, methods of manufacturing said systems and methods of using thesystems to manufacture cellular therapeutics.

WO 2005/035728 describes a system with a gas permeable portion forculturing cells. This device is available from Wilson Wolf under thebrand G-Rex Technology (please see the websitewww.wilsonwolf.com/page/show/67596). The main benefit of the system isthat it allows nutrients and gases to be provided to growing cells in away so that the cells can be continuously grown for periods of up to 14days without any further intervention, in particular: the gas permeablemembrane allows exchange of CO₂ and O₂ and the arrangement allows largevolumes of media to be employed which provides all the nutrientsnecessary for growth. The arrangement is shown in FIG. 1.

The main limitation of the G-Rex technology is that it is an openmanufacturing system that does not allow the inoculation and harvestingof cells and addition of nutrients without exposure to the externalenvironment. Since cellular therapeutics cannot be sterilised postproduction, their manufacture has to occur under aseptic conditions.Thus the “open” processing steps during which the product is exposed tothe external environment have to be performed under a laminar air flowcabinet which is operated in a clean-room classified according to EU-GMPclassification class B (US Fed. Std. 209e class 10,000, ISO 14644-1class ISO7) to prevent contamination of the product with microbes andparticles. Facilities with such clean-room technology, for openprocessing, are expensive to build, operate, maintain and monitor.

Equally important is the fact that the open operation steps require thatonly one product can be handled at a time in the same clean-room spaceto minimise the risk of cross-contamination, hence, the productthroughput is limited and this manufacturing system requires multipleclean-rooms and production teams to operate in parallel to achieve highvolumes of production output. For small and medium size companies aswell as hospitals, the capital investment and labour costs are verysignificant on a per unit basis of production.

To address this problem the present inventors have provided a modifiedsystem for the culture of cells that allows aseptic manufacturing ofcells for use in therapy, without the need for a clean-room environmentbecause no open processing steps are required.

SUMMARY OF THE INVENTION

Thus the present invention provides a closed system suitable foraseptically culturing therapeutic cells comprising

-   i) a vessel comprising:    -   a gas permeable portion suitable for supporting cell growth and        allowing delivery of gases to the cells during culture, and    -   at least one wall adjoined to a base, wherein said vessel        defines an internal volume and said vessel is adapted to contain        a requisite volume of medium to support cells during culture,-   ii) a vent comprising a conduit defining an interior orifice and an    exterior orifice distal therefrom in fluid communication with each    other, wherein the conduit extends from the exterior of the closed    system through a structural feature of the system, optionally    extends into the internal volume of the vessel, and terminates in    the internal volume with the interior orifice, wherein the interior    orifice is located in the internal volume such that during filling    and emptying of liquid media and/or cells it is not susceptible to    blockage by liquid or cells,    -   wherein the exterior orifice is adapted to connect to an aseptic        filter thereby allowing gases to pass through the filter into        the vessel or out of the vessel, as required,-   iii) a port adapted to allow introduction of fluid and cells    aseptically into the vessel,    -   a port adapted to fluid and cells to exit the system without        exposing the system to the external environment and adapted such        that cells grown therein may exit the system under gravity when        the system is orientated to put the cells fluid communication        with the exit port and the latter is open.

The system according to the present invention has significant benefitsfor the commercial manufacturing of cells for use in therapy. Inparticular it is flexible and adaptable, requires low capital investmentinto manufacturing space, and is robust and easily manufactured. It alsoeliminates open processing steps and therefore further reduces the riskof contamination of the product with pathogens and particles. Further,the present system is believed to meet a currently unmet need, andrepresents a real step forward in reducing the need for expensiveclean-room facilities for the manufacture of therapeutic cells.

Advantageously, the present invention allows the transformation ofexisting open system technology, such as the G-Rex system, to provide aclosed system that allows the inoculation and harvesting of cells andthe provision of nutrients without exposure to the external environment.

Alternatively, a bespoke system according to the present disclosure maybe manufactured specifically for culturing cell therapeutics, forexample a system shown in FIG. 7 or 8.

The present disclosure also extends to methods of manufacturing thesystems described herein and also use of the systems to culture cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of the commercially availableG-Rex system for cell culture available from Wilson Wolf.

FIG. 2 is a diagrammatic representation of a vessel comprising a lidwith a vent and port co-located therein.

FIG. 3 is a diagrammatic representation of a unitary vessel according tothe present disclosure comprising a vent and port co-located in a wallof the unit.

FIG. 4 is a diagrammatic representation of a unitary vessel according tothe present disclosure comprising a vent and a port located opposingeach other.

FIG. 5 is a diagrammatic representation of a unitary vessel of therepresent disclosure wherein the vent and port are arrangedperpendicular relative to each other.

FIG. 6 is a diagrammatic representation of a vessel comprising a lidwith a vent and port co-located therein, wherein the vent extends downinto the media towards the gas permeable layer. The interior orifice iscovered by a liquid impermeable membrane, such as a gas permeablemembrane to prevent media entering the vent.

FIG. 7 is a diagrammatic representation of a bell shaped unitary vesselaccording to the present disclosure comprising a vent and portco-located in a wall of the unit.

FIG. 8 is a diagrammatic representation of a bell shaped unitary vesselaccording to the present disclosure comprising a vent and portco-located in a wall of the unit and orientated to allow access of themedia and cells to the exit port.

FIG. 9 is a diagrammatic representation of how a system according to thedisclosure can be filled and harvested.

FIG. 10 is a diagrammatic representation of a system according to thepresent disclosure

FIG. 11 are detailed drawings of a system as shown in FIG. 10

FIG. 12 is a diagrammatic representation of how to orientate the systemto harvest cells cultured therein.

DETAILED DESCRIPTION OF THE INVENTION

A closed system as employed herein allows entry and exit of materialsincluding liquids, cells and gases without exposure to the externalenvironment.

A closed system according to the present disclosure can nevertheless beemployed as an open system if the end user decides not followappropriate protocols when introducing or extracting material from thesystem. However, the systems disclosed herein are arranged and adaptedto be suitable for use as a closed system.

Culturing cells as employed herein is intended to refer to expandingand/or differentiating cells in vitro.

Cell expansion as employed herein is refers to increasing the number ofthe target cells in a population of cells.

WO 2005/1035728 incorporated herein by reference describes how toprepare a gas permeable vessel. In one embodiment silicone gas permeablematerial is employed.

In one embodiment the base supports incorporates the gas permeablematerial.

In one embodiment the base substantially consists of gas permeablematerial i.e. the gas permeable material layer and the base are in factthe same entity.

In one embodiment the gas permeable layer is located in a wall or otherstructure feature of the vessel.

In one embodiment substantially all of the vessel is prepared from a gaspermeable material, for example with sufficient structural strength toretain the contents during culturing.

In one embodiment the gas permeable membrane has a surface area of, forexample 5 to 200 cm², such as 5 to 100 cm², in particular 10, 30 or 50cm², such as 10 cm².

A base as employed herein is a structural element of the vessel.

When cells are in the process of being cultured generally the systemswill be orientated (or stood) on the base, for example the base is flator substantially flat. Even if the system is not stood on the base, toallow air to circulate, then during culturing the base may represent thelowest part of the system.

Generally during the culturing process the cells tend to settle on andbe supported by the internal surface of the gas permeable layer, forexample in a plane which is parallel to the plane of the base.

The base in the context of the present disclosure, for example can beunderstood by reference to the devices shown in FIGS. 5 to 8, whichshows the base (1). Clearly the gas permeable layer in the base musthave access to the environmental gases for the system to function. Thusthe base may be raised above the surface on which the system is stood orlocated, to ensure access to the external gaseous environment.

Adjoined thereto as employed herein is intended to refer to the factthat one element is attached to another.

The vessel employed will generally be rigid or substantially rigid orresiliently deformable but not permanently deformable. However thevessel may comprise portions of flexible or deformable materials. Theseflexible materials may include the type of materials employed in themanufacture of infusion bags.

In one embodiment the vessel and substantially all the structuralelements thereof are rigid.

The vessels and systems of the present invention may be provided in awhole range of shapes and sizes, for example derived from a cube, box,cylinder, cone or pyramid. However, usually at least one area or side ofthe shape will be adapted for accommodating vents, port or ports and/orother elements of the system. Pure shapes may be used but will notgenerally be employed because the shape will usually be adapted toprovide a bespoke vessel for the intended purpose. For example a coneshape may be adapted to provide a frusto-conical shape comprising a baseand curved wall and a top wall or lid. The top wall or lid mayaccommodate the port(s) and/or vents.

In one embodiment the vessel is associated with or comprises are-sealable lid, wherein the vessel and the lid together form the closedsystem. A re-sealable lid can be illustrated by reference to FIG. 1which shows a device comprising a screw lid, which is one type ofre-sealable lid.

In one embodiment, the attachment of the lid to the vessel is byscrewing the lid through thread etched in the vessel thereby allowing aseal to be created which protects the contents therein fromcontamination from microbes and particles in the external environment.

In one embodiment a system according to the present disclosure can beprovided by modifying an existing system, for example the G-Rex unit canbe modified by incorporating a vent and one or two ports, to which othercomponents of the manufacturing system can be aseptically connected. Inone embodiment the vent and port or ports are incorporated into the lidof a G-Rex system. This provides a cost-effective way of producing aclosed system for culturing cell therapeutics, which can be operatedoutside of a laminar air flow cabinet in a clean-room classifiedaccording to EU-GMP class D (US Fed. Std. 209e class 100,000, ISO14644-1 class ISO8) since the product is never exposed to the externalenvironment. This greatly facilitates the aseptic manufacturing of thecellular therapeutic in conformance with regulatory requirements.

In one embodiment there is provided a lid according to the presentinvention for a manufacturing system or vessel according to thedisclosure, such as a G-Rex system, in particular said lid comprising avent and/or port as discussed herein or fitting to accommodate same.

In one embodiment the lid, vent and/or ports are unitary, for examplemoulded.

In one embodiment the lid, vent and/or ports are the same material.

In one embodiment the lid, vent and/or ports are distinct materials.

In one embodiment the lid, vent and/or ports are one material and thevessel onto which the lid is adapted to fit is the same material.

In one embodiment the lid, vent and/or ports are one material and thevessel onto which the lid is adapted to fit is a distinct material.

In one embodiment the lid, vent and/or ports are distinct materials andthe vessel onto which the lid is adapted to fit is a material employedin the lid or vent/port.

In one embodiment the lid, vent and/or ports are distinct materials andthe vessel onto which the lid is adapted to fit is a further distinctmaterial.

In one embodiment the a hard synthetic material suitable for use inaseptic manufacturing as described herein, such as polycarbonate, isemployed to manufacture one or more of the above components.

It may be advantageous from a GLP and regulatory perspective for thevessel, lid, vents and ports to be the same material.

In one embodiment the vessel is unitary in nature in that it defines acomplete unit without a removable or re-sealable element, such as a lid.Unitary vessels may, for example be moulded in one piece but unitary asemployed herein is not a reference to how the vessel is made but ratheris a description of the function of the vessel and in particular that noadditional structural elements, such as a lid, are required to seal thevessel.

In one embodiment there is provided a bell arrangement (FIGS. 7 and 8)wherein a structural feature opposing the base provides a concavesurface in the interior volume. Shapes such as bells are advantageousbecause the number of internal corners in the internal volume areminimised which may maximise the recovery of cells possible. The presentdisclosure also extends to alternative shapes which are suitable forperforming this function, in particular wherein the locations capable oftrapping cells are minimised by using “rounded surfaces”.

Thus in one embodiment the vessel is arranged to funnel cells to theexit port when the system is situated in the appropriate orientation. Inone embodiment the internal shape is adapted to facilitate drainage froman exit port when appropriately orientated.

The systems according to the present disclosure are arranged to allowremoval of liquid and cells under gravity, when arranged in the requiredorientation. Removing the cells under gravity is advantageous because itis simple, efficient and cost-effective. Nevertheless this process maybe augmented by employing a vacuum, increasing the internal pressure ofthe system (referred to herein as overpressure) or pumping (such as aperistaltic pump). These technologies are well known and may be employedin combination with the system by attachment of the pump, vacuum or gasinput to create an over-pressure to a relevant port or vent, asappropriate.

If desired the liquid and cells may be removed by pumping, vacuum orover-pressure without the assistance of gravity, even though the systemis designed to be suitable for removal of the liquid or cells gravity.

A structural element as employed herein is intend to refer to abase,wall, lid or other structural feature of the vessel that performs afunction such as supporting, retaining shape and volume, holding or thelike. A structural element does not refer to appendages to the vesselsuch as accessories, in particular ports, vents, gaskets and the like.

Unless the context indicates otherwise interior and internal areemployed interchangeably herein.

Unless the context indicates otherwise exterior and external areemployed interchangeably herein.

Interior volume and interior space are employed interchangeably herein.

In one embodiment the requisite volume of media which the vessel isadapted to contain is an amount that does not block the vent duringculturing the cells and removal of the liquid and cells. In oneembodiment the volume of media is 50% or less of the internal volume,such as 45%, 40%, 35%, 30%, 25%, 20% 15%, 10% or less. When the vesselis filled to this level then it may be appropriate to remove thecontents under gravity.

In one embodiment the maximum requisite volume of media is 15 to 30 ml,such as about 20, 21, 22, 23, 24, 25 or 26 ml.

“Not susceptible to blockage by media and cells” as employed herein isintended to refer to the fact that the elements are arrangement tominimise access of the cells and/or media to the feature, such that ithas a reduced propensity to becoming blocked such that is cannot performits function.

In one embodiment the volume of media is 50% or more of the internalvolume, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In thisembodiment the maximum requisite volume of media in a system such as theG-Rex 10 is 30 to 40 ml, such as about 31, 32, 33, 34, 35, 36, 37, 38 or39 ml. When the vessel is filled to this level then it may beappropriate to employ a “pump” or other system described above to assistin removing the contents. The vent may be cleared of any media or cellslocated therein by the “pumping”/foreed extraction process and thus thevent may become “blocked” if this system is emptied under gravitationforces only.

In one embodiment the ratio of the numeric values of the gas permeablearea to the volume of the medium employed in the systems is in the range1:1 to 1:5 respectively, for example 1:2, 1:3 or 1:4, such as 10 cm² gaspermeable area to 20 ml volume which gives a ratio of 1:2.

The vent comprising a conduit defining an interior orifice and exteriororifice is essentially a pipe connecting the interior of the system tothe exterior. This vent allows balancing of pressure during filling thevessel by allowing gases to exit. During the cell emptying process thevent allows gases to enter the internal volume to fill the void thatwould be created by removal of the liquid and cells, thereby allowingfree fluid communication of the liquid and cells to the external portusing gravity or pressurized systems.

The vent terminates into the internal volume is intended to refer to thefact that the vent has access to the internal volume and the interiororifice may be located in a structural feature of the vessel such as awall, provided that the vent is in fluid communication with the internalvolume and that the vent is arranged such that it is generally notsusceptible to blocking during harvesting of the cells, when the mediaand cells are less than 50% of the volume of the vessel.

Extends into the internal volume is intended to refer to part of thevent conduit protruding into the internal space.

In a preferred embodiment at least part of the vent physically extendsand protrudes into the internal volume like an appendage anchored in thestructural feature.

In one embodiment the portion of the vent terminating in the internalorifice does not terminate in the same plane as the structural elementthrough which it is supported, that is to say the conduit will generallypass through an structural element in which it is supported and extendsinto the space defined by the internal volume, for example terminatingcentrally in the volume as shown in any one of FIGS. 2, 3, 5, 6, 7 and10 to 12.

Located centrally as employed herein is intended to refer to the factthat part of the vent extends from the structural feature of the vesselin which it is housed and into the space of the internal volume and isnot intended to be an absolute reference to the centre of the space perse. This feature has the benefit that during filling of the vessel withmedia or during harvesting of cells (in particular if the vent isco-located with the port for entry/exit) then exposure of the internalorifice to liquid is thereby minimised.

In one embodiment the vent is arranged to terminate in the volumetriccentre of the internal volume of the vessel.

Volumetric centre as employed herein is intended to refer toapproximately the centre of the three dimensional space defined by theinternal volume.

In one embodiment the conduit of the vent extends between 5 and 35 mm,such as about 30 mm from the underside of the lid or other structuralfeature of the vessel or alternatively up from the base.

In one embodiment the interior orifice of the vent is arranged not tocontact the media contained in the vessel during use, in particular whenthe media and cells are less than 50% of the volume of the vessel, suchthat the orifice and vent are not susceptible to being blocked by liquidor cells.

However the vent may be arranged such that it extends into the mediacontained in the vessel during use. The function of the vent may befacilitated by employing a liquid impermeable membrane, such as a gaspermeable membrane over the internal orifice and/or employing a liquidnon-return valve in the vent.

In one embodiment the vent may comprise a liquid non-return valve.

In one embodiment the internal orifice may be protected by a liquidimpermeable membrane, such as a gas permeable membrane.

Optionally the vent may comprise a valve to control follow of gases, ifdesired.

The vent is arranged to be capable of attaching to a sterile filter suchas a 0.2 micron filter that prevents airborne microbes and particlesentering the internal volume of the vessel and contaminating the system.Generally the filtering device or element will be attached to theexternal orifice of the conduit forming the vent, as shown in FIGS. 2 to6 and 9.

The attachment may be direct or via a coupling means, such as tubing.

In one embodiment the sterile filter is secured to the vent by a fixingmeans such as a leur lock.

The vent may be prepared from any suitable material and may be mouldedin the structural feature that supports it i.e. integral thereto.

In one embodiment the vent may simply comprise flexible tubing extendingthrough the structural feature that supports it.

In one embodiment the vent comprises an exterior portion extendingoutside the closed system which is suitable for connection to flexibletubing.

-   Suitable flexible tubing is available in many different forms, for    example:    -   a translucent tubing which contains no plasticizers, latex and        vinyl acetate and is also free of animal products, or    -   transfusion tubing, for example PVC transfusion grade tubing        such as 4 mm OD PVC transfusion grade tubing.-   In one embodiment the tubing is silicon.

The tubing employed is such that it can be connected aseptically tocomponents also containing a tube using a sterile tube welder, forexample available from Terumo Medical Corporation.

The vent can be located in any suitable location on the vesselincluding, for example in a structural feature such as a wall, the base,a lid, for example in one embodiment the vent extends through the gaspermeable layer, which as described in detail above and may be locatedin the base of the vessel.

In one embodiment the vent is located centrally on a structural featureof the vessel, that is to say not proximal to an edge of a wall, base orlid.

In one embodiment the vent is located approximately along a central axisof the vessel, for example through the centre of a lid or wall and mayextend to the centre of the internal volume. Centre as employed hereinis intended to refer to approximately the midpoint of the space orfeature.

Suitable material for the port or ports when moulded include, forexample the same materials as the vessel (such as thermoplastics, inparticular polycarbonate). In one embodiment a moulded port or portsis/are designed such that the port or ports are suitable for connectingto tubing, for example as shown in FIG. 6 where the tubing slides ontothe vent or port portion which is moulded.

In one embodiment the port or ports are provided as tubing, such asflexible tubing, extending through a structural feature of the vesseland/or system. In this embodiment the flexible tubing may need to besealed to the structural feature by a gasket.

Tubing for use with or as a port include tubing described supra that canbe used in sterile connecting devices for the aseptic connection ofexternal components like e.g. sterile infusion bags.

The port or ports can be located in any suitable location on the vesselincluding for example structural features such as a wall, the base, alid.

In one embodiment there is provided a separate entrance port and aseparate exit port.

In one embodiment there is provided one port which functions as anentrance and exit port.

In one embodiment the vent is co-located with a port or ports, forexample in a wall or lid.

The benefit of co-locating the vent and port or ports in a structuralelement opposite to the base, such as a lid or wall, is that nomodifications to the vessel side wall are required. This is advantageousbecause vessels without features in the side walls can be placed inclose proximity to each other occupying minimal space. This also allowsefficient manufacture of the vent and port or ports within a singlecomponent of the vessel.

The benefit of co-locating the vent and port or ports in the lid meansthat no further modification of the commercially available vessel isrequired to convert it from an open into a closed system.

In one embodiment the port employed for cells to exit is locatedproximal to a structural element, such as wall, the edge of a lid or thelike. The inventors have found by locating the exit port by an edge, ofa wall or structural element of the vessel then a more efficientrecovery of cells is obtained. An example the port located proximal to astructural feature is shown in FIGS. 2 to 12.

Having the port or ports to the side of the lid or other structuralfeature ensures that maximum recovery of liquid and cells is achieved.Earlier prototypes had the port in the centre of the lid, and due to thesurface tension of the liquid, it was determined that 0.5 ml-0.8 ml ofliquid was retained in the vessel. When the port was moved to the side,and flush with a gasket, the retention was reduced to 0.1 ml.

In addition locating the port or ports off-centre allows the vent to belocated centrally, which in at least some embodiments may minimise theexposure of the vent to blockage by liquid.

Thus in one embodiment the port or ports, in particular the exit port isnon-coaxial with a central axis of the internal volume of the vessel.

In the cross-sectional figures shown herein a double line represents awall or barrier or similar which is closed. However a single line is notclosure but is present to show the shape of the feature. Thus where asingle line is shown at the end of a vent or port, the same is open.

FIG. 2 shows a gas permeable layer (2), which forms the base (1) ofvessel (4), which comprises a lid (8) co-locating a vent (6) and a port(5), wherein said vent comprises a conduit defining an exterior orificecapable of supporting a sterile filter said conduit extending throughthe lid and extending centrally into the interior volume defined by thevessel and terminating in the interior orifice and wherein said interiororifice is covered with a liquid impermeable membrane (9) or a liquidnon-return valve (10). In use the vessel contains media and cells whichsettle on the interior surface of the gas permeable layer.

FIG. 3 shows a similar arrangement to that of FIG. 2 but ischaracterised by the vessel being a single unit without a re-sealablelid. The port (5) and vent (6) are co-located in a wall (a structuralelement) of the vessel. The conduit of the vent extends as a protrusioninto the internal volume of the vessel.

FIG. 4 shows a similar arrangement to FIG. 3 but wherein the port (5)and vent (6) are arranged to oppose each other.

FIG. 5 shows a similar arrangement to FIGS. 3 and 4 but wherein the port(5) and the vent (6) are arranged perpendicular to each other.

FIG. 6 shows a gas permeable layer (2), which forms the base (1) ofvessel (4), which comprises a lid (8) co-locating a vent (6) and a port(5), wherein said vent comprises a conduit defining an exterior orificecapable of supporting a sterile filter said conduit extending throughthe lid and extending centrally into the interior volume, defined by thevessel, towards the base and into the media (3). The interior orifice isprotected by a gas permeable membrane (9) which prevents blockage of thevent by liquid.

FIG. 7 shows a unitary bell shaped vessel (4) arranged with the port (5)(which has a dual function of the entrance and exit port) and the vent(6) co-located in a structural feature of the vessel opposing the basecomprising a gas permeable layer. In this Figure the vent (6) and port(5) are shown with the exterior portions connected to flexible tubing.

FIG. 8 shows a system of FIG. 7 orientated to put the liquid media andcells in communication with the exit port (5), wherein the cells mayexit the port under gravity. The dimensions of the Figure herein are byway of example only and are not intended to be limiting.

Sterile containers, such as infusion bags, containing materials such asa media and/or cells can be welded to an entrance port or tubingconnected thereto using known technology to introduce media asepticallyinto the system. This is illustrated diagrammatically in FIG. 9. Sterilewelding techniques and technology are well known in the industry andwill not be discussed further here.

Similarly empty containers such as sterile infusion bags can be weldedonto an exit port, or tubing connected thereto, to remove the cells fromthe vessel aseptically, also illustrated in FIG. 9.

Both of these methods of introducing and removing materials from thesystem aseptically are very convenient, robust and practical.

Allowing cells to be removed (harvested) under gravity by changing theorientation of the vessel by 10 to 350 degrees, for example 45 degreesor greater such as 90 or 180 degrees is very easy and efficient becauseit does not require specialised equipment and needs minimal space andexpertise.

In one embodiment the exit port does not extend above a structuralfeature in which it is situated into the internal volume more than 0.15mm. The later ensures maximum recovery of cells.

The port or ports may be fitted with a valve and/or membrane or the tocontrol flow of materials therethrough.

Suitable gaskets and seals may be required depending on the exactconstruction of the system.

A suitable material for manufacturing the vessel and structural elementsthereof will generally be required to be compliant with one or more ofthe standards: EU Food Approval: EU Directive 2002/72/EC; USP <88>,biological reactivity test in vivo, class VI; USP <87>, biologicalreactivity test in vitro; USP <661>, physicochemical test—plastics; EPmonograph 3.2.2, plastic containers & closures for pharmaceutical use;Biological tests according to ISO 10993—external communicating devices;for indirect blood contact for a prolonged period.

In one embodiment the vessel and/or structural elements of the systemsare manufactured from a suitable thermoplastic such as polycarbonate.

The ports and vents may be incorporated into the system by thermalsoldering, especially when a closed system is being created from acommercially available product, such as G-Rex.

In one embodiment a modified lid will be moulded in one piece withappropriate connections for tubes and/or filter, which will be attachedafterward.

In one embodiment the features and structural functionality, such asports and vents, will be created when moulding the vessel.

Structural functionality as employed herein is intended to refer tofeatures which perform a function, for example as a vent or port and/orwhich is suitable for attaching accessories to, such as tubing or thelike.

The systems according to the disclosure may be gamma irradiated forsterilisation.

Generally the system will be delivered to end users in a sealed bag in asterile form, for example along with a certificate of sterility. Asystem in this form will generally have a shelf-life of about 1 year ormore.

There is also provided a process of manufacturing a system according topresent disclosure comprising the step of moulding the vessel, andoptionally fitting the ports and vents thereto.

In one embodiment the holes to accommodate a vent and/or ports arecreated by thermal probes rather than by drilling, thereby minimisingthe amount of contamination generated and ensuring the final product issuitable for use to prepare a therapeutic product.

In one embodiment further components such as gaskets and/or valvesand/or filters are fitted in the manufacturing process.

In one embodiment the manufacturing process comprises the further stepof sterilising the system, for example using gamma irradiation andaseptically sealing the unit in one or multiple bags or containers.

In one embodiment one or more steps of the manufacturing process areperformed in a clean-room complying with standards to EU-GMP class D (USFed. Std. 209e class 300,000, ISO 14644-1 class ISO8). In one embodimenteach system together with filters, gaskets, tubes and connections andbefore sterilisation the assembled system will be subject to a pressurehold test with compressed air according to methods known to thoseskilled in the art, for example pressure testing of 0.5 bar±0.05 bar forgreater than 2 mins. During this time, the pressure will not drop fromits recorded start value by less than 0.05 bar.

In one embodiment there is provided a method of modifying an existingcell-culture system to provide a closed system according to theinvention. The modification may employ one or more of the manufacturingsteps defined above.

In one embodiment a vessel according to the disclosure, such as amodified G-Rex vessel and/or a vessel with a gas permeable membrane ofabout 10 cm² will typically be filled to 10 to 25 ml final volume withcells and culture medium, and will be incubated at 37° C. until thecells are ready for harvesting. Having this the vessel may be filledwith up to 40 mls as discussed above.

Thus a method of introducing cells aseptically to a closed systemaccording to the disclosure followed by incubating the cells at anappropriate temperature and for an appropriate period is provided.

In a further aspect, a method of aseptically harvesting therapeuticcells from a closed system according to the disclosure by asepticallyjoining a receptacle to an exit port of the system and harvesting thecells into receptacle under gravity is provided.

In a further aspect, a method of aseptically harvesting therapeuticcells from a closed system according to the disclosure by asepticallyjoining a receptacle to an exit port of the system and harvesting thecells into receptacle employing a pump is provided.

In one embodiment the receptacle into which the harvested cells aretransferred is a 600 ml bag, sometimes referred to in the field as a“transfer pack”.

In one embodiment after harvesting the cells are washed, for examplewith a solution comprising human serum albumin, saline or similar.

In one embodiment the cells are washed manually.

In one embodiment the cells are washed employing an automated system,for example a Sepax® system available from Biosafe.

In one embodiment, for example after washing, the cells are counted.

In one embodiment, for example after counting, a therapeutic amount ofcells (i.e. one or more doses of cells) is selected.

In one aspect the harvested cells are enclosed into a suitablecontainer, for example an infusion bag, optionally along with one ormope pharmaceutically acceptable preservatives or excipients, forstorage.

Suitable excipients include DMSO, for example 10% DMSO.

In one embodiment the container is transported to the location of apatient.

In embodiment the content of the container is administered to a patientparenterally, particularly intravenously.

In one aspect the invention relates to equine training equipmentsubstantially as defined herein.

In the context of this specification “comprising” is to be interpretedas “including”.

Aspects of the invention comprising certain elements are also intendedto extend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements.

We claim:
 1. A closed system suitable for the aseptic culture oftherapeutic cells comprising: (i) a vessel comprising: a gas permeableportion suitable for supporting cell growth and allowing delivery ofgases to the cells during culturing, and at least one wall adjoined to abase, wherein said vessel defines an internal volume for containing arequisite volume of medium to support a cell culture, (ii) a lid (iii) avent in the lid which allows balancing of pressure during filling andemptying of the vessel said vent comprising a conduit defining aninterior orifice and an exterior orifice, wherein the conduit extendsfrom the exterior of the closed system through lid and extends into theinternal volume of the vessel and terminates therein with the interiororifice, and wherein the interior orifice is arranged not to contact themedium in the vessel for all orientations of the vessel including whenthe system is inverted, or wherein the interior orifice is covered witha liquid impermeable membrane or a liquid non-return valve, and (iv) anexit port located in the lid which allows cells grown in the vessel toexit the system under gravity when the system is orientated to put thecells in fluid communication with said exit port; and wherein said ventconduit extends further into the interior volume than said exit portdoes.
 2. A system according to claim 1, wherein part of the vessel isarranged to funnel cells to the exit port when the vessel is in anappropriate orientation.
 3. A system according to claim 1, wherein theexit port is non-coaxial with a central axis of the internal volume ofthe vessel.
 4. A system according to claim 1, wherein the exit port islocated in the lid and the port extends 0.15 mm or less into theinternal volume.
 5. A system according to claim 1, wherein the conduitof the vent extends between 5 and 35 mm into the internal volume of thevessel.
 6. A system according to claim 1, wherein the conduit of thevent extends 30 mm from the lid into the internal volume of the vessel.7. A system according to claim 1, wherein said vent and said exit portare located opposing the base.
 8. A system according to claim 1, whereinthe system is arranged to provide aseptic emptying under gravity.
 9. Asystem according to claim 1, wherein the interior orifice is arrangednot to contact the medium in the vessel when the system is emptied undergravity by inversion of the system.
 10. A system according to claim 1,wherein the interior orifice of the vent is covered with a liquidimpermeable membrane or a liquid non-return valve.
 11. A systemaccording to claim 1, wherein the system is emptied under gravity bychanging the orientation of the system by more than 90 degrees.
 12. Asystem according to claim 1, wherein the vent conduit extends toapproximately the midpoint of internal volume of the vessel.
 13. Asystem according to claim 1, wherein the vent is located centrally onthe lid and the exit port is located to the side of the lid.
 14. Asystem according to claim 1, wherein one port is an entrance and also anexit port.
 15. A system according to claim 1, wherein the systemcomprises a second port to provide aseptic introduction of fluids andcells into the vessel.